Having paid a great effort to literature and experiment, we have engineered a brand new system combining aptamer and Cas12a to detect antibiotics, which features two key rules when designing the part. The data showed that the system worked extremely successfully.
To stop the antibiotic resistance worsening, thousands of environmental inspectors in China are sent out to measure antibiotic residues in the environment, which is important for antibiotic-abuse surveillance. To make the process more convenient, we want to develop a portable and rapid-response method for on-site antibiotic-residue detection. After paying a great effort into the literature, we found that aptamer is a great tool to detect small molecules including antibiotics1. And aptamer for kanamycin, a kind of antibiotics, has been created and reported to have the strong binding activity 2,3. Aptamer is a kind of nucleic acid that is similar to antibody but cheaper and more flexible. However, it has several other advantages, one of which is its ability of structure switching upon the recognition of the specific target. Using screening methods like SELEX, specific aptamers with strong binding activity will be selected out. Take kanamycin aptamer as an example, upon the binding of kanamycin, it would form a G-quadruplex.
Now we have the tool for antibiotic binding. But aptamer itself can not give out signals, how could we use it in antibiotic measuring? To achieve this, we need to combine aptamer with our Cas12a reporting module. We immediately head for the literature for inspiration. Luckily, we found a solution can probably be the bridge to combine aptamer and Cas12a-reporting module4. They found an oligonucleotide which could base pair with the aptamer. It is not until the existence of small molecules that the oligonucleotide can be recognized by Cas12a and give off signal. Inspired by this work, we set about developing a novel system targeting antibiotic residues combining these two powerful tool. Please refer to the design page for details.
Figure 1.Workflow of the system in the literature, adapted from [4].
Recombinase Polymerase Amplification (RPA)
Unsurprisingly, the results turned out to be extremely good. We did orthogonal experiments concerning the concentrations of aptamer-activator DNA complex and kanamycin. We concluded that, as the concentration of kanamycin increased, the fluorescence intensity increased as well (Figure 3a, b).
Figure 3. The characterization results of CESAR-1 system. (a) The fluorescence intensity over different concentrations of kanamycin, i.e., 0, 170, 340. The concentration of aptamer-activator DNA complex is 12.5nM. RFU refers to relative fluorescence unit. (b) The bar graph of fluorescence intensity at 166min.
To summarize, we designed a brand-new element for aptamer to combine with Cas12a detection system. We set out from our needs, created the designed based on literature information, and finally did experiment to successfully tested it. We are proud of our achievement, and it is a great pleasure that we can provide to future iGEM teams with our independently developed components!
Reference
1.Dunn, M. R., Jimenez, R. M., & Chaput, J. C. (2017). Analysis of aptamer discovery and technology. Nature Reviews Chemistry, 1(10), 76.
https://doi.org/10.1038/s41570-017-0076
2.Ma, X., Qiao, S., Sun, H., Su, R., Sun, C., & Zhang, M. (2019). Development of structure-switching aptamers for kanamycin detection based on fluorescence resonance energy transfer. Frontiers in Chemistry, 7(FEB), 1–10.
https://doi.org/10.3389/fchem.2019.00029
3.Xing, Y.-P., Liu, C., Zhou, X.-H., & Shi, H.-C. (2015). Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay. Scientific Reports, 5, 8125.
https://doi.org/10.1038/srep08125
4.Xiong, Y., Zhang, J., Yang, Z., Mou, Q., Ma, Y., Xiong, Y., & Lu, Y. (2020). Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets. Journal of the American Chemical Society, 142(1), 207–213.
https://doi.org/10.1021/jacs.9b09211